用于口腔骨组织修复藻酸钙凝胶的制备及生物学性能研究

发布时间：2018-02-09 00:48

本文关键词： 海藻酸钙水凝胶人牙周膜细胞纳米银生物安全性能成骨能力　出处：《南方医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的本课题旨在制备有助于口腔牙周骨组织再生的可载药海藻酸钙凝胶及其纳米复合材料,检测材料物理性能,同时检测体内和体外生物学性能,最后研究复合材料的抗菌能力。为后期进行3D打印复合支架的研究和其临床应用奠定基础。材料与方法材料:海藻酸钠粉末、氯化钙粉末、牛血清白蛋白、高糖培养基、胎牛血清、青链霉素抗菌剂、胶原酶、PBS溶液、二甲基亚砜、MTT检测试剂盒、成品纳米银、LB营养培养基、苏木素、伊红、二甲苯、冰冻切片机、倒置显微镜、Micro-CT 机。方法:离子交联法制备海藻酸钙水凝胶后进行成分检测;使用BCA比色法测量材料蛋白释放量;监测干重与湿重进行吸水率测定;分别测量3天、7天、14天、28天及56天时预定时间点的干重及湿重,分析材料降解情况;进行牙周膜细胞原代的培养,使用免疫组化染色进行鉴定;MTT(四唑盐)法检测人牙周膜细胞(hPDLCs)的增殖及海藻酸钙水凝胶对细胞毒性;RT-PCR检测材料对hPDLCs炎症相关因子表达以及骨髓基质干细胞成骨相关基因表达的影响;茜素红染色法检测矿化能力;通过肉汤稀释法测量纳米银颗粒最低抑菌浓度;离子交联法制备海藻酸钙/纳米银复合水凝胶材料并对其成分分析;采用抑菌圈法检测纳米银复合海藻酸钙水凝胶对金黄色葡萄球菌的抑菌作用;HE染色观察海藻酸钙水凝胶对软组织初期炎症反应;制备下颌骨缺损模型;采用Micro-CT对海藻酸钙水凝胶骨修复能力评价。所有实验均重复三次。统计学分析采用SPSS 19.0统计软件对数据(x±S)进行单因素方差分析(one-wayANOVA)分析。若方差齐,则用Dunnett和Bonferronili对数据进行两两比较分析;若方差不齐,则使用Dunnett' s分析,p0.05表示具有统计学差异。结果成功制备可载药注射型海藻酸钙水凝胶;吸水率测定显示海藻酸钙水凝胶显示出良好的吸水性能;降解实验结果表明25 mg/mL浓度的海藻酸钙水凝胶其降解时间与骨组织再生成时间更为匹配;三种浓度海藻酸钙水凝胶均处于1级,可认为材料对于(?)牙周膜细胞无毒性,生物安全性能良好。海藻酸钙水凝胶及PLA支架材料均促进牙周膜细胞炎症因子的表达;海藻酸钙水凝胶共培养后的细胞形成的矿化结节比空白组及PLA组明显增多;海藻酸钙水凝胶促进成骨相关因子表达;成功制备海藻酸钙水凝胶载纳米银材料;复合了纳米银后材料具备抑菌作用;海藻酸钙水凝胶导致的软组织炎症反应轻,且与组织结合良好;海藻酸钙水凝胶对骨组织缺损诱导再生效果较PLA材料好。结论成功构建可载药海藻酸钙材料,体内及体外实验验证其应用于口腔组织中具有良好生物学性能;具有促进细胞骨向分化的能力;25 mg/mL的藻酸钙凝胶效果最好;成功制备出的海藻酸钙水凝胶载纳米银材料具有抑菌性;有助于预防伤口感染。
[Abstract]:Objective to prepare calcium alginate gel and its nanocomposites which can be used for oral periodontal bone regeneration, and to test the physical properties and biological properties in vivo and in vitro. Finally, the antibacterial ability of composite materials was studied. It laid a foundation for the study of 3D printing composite scaffold and its clinical application. Materials and methods: sodium alginate powder, calcium chloride powder, bovine serum albumin, high sugar medium, Fetal bovine serum, streptomycin antimicrobial agent, collagenase PBS solution, dimethyl sulfoxide MTT assay kit, nano-silver lb nutrient medium, hematoxylin, eosin, xylene, frozen slicer, Methods: the components of calcium alginate hydrogel were prepared by ion crosslinking method, the protein release was measured by BCA colorimetry, the water absorption was measured by dry weight and wet weight. The dry and wet weights were measured at 3 days, 7 days, 14 days, 28 days and 56 days, respectively. The degradation of the materials was analyzed, and the primary culture of periodontal ligament cells was carried out. Immunohistochemical staining was used to detect the proliferation of human periodontal ligament cells (hPDLCs) and the expression of inflammatory factors related to hPDLCs and osteogenic phase of bone marrow stromal stem cells (BMSCs) were detected by reverse transcription-polymerase chain reaction (RT-PCR) of calcium alginate hydrogel. The effect of gene expression; Alizarin red staining method was used to detect mineralization ability, the minimum inhibitory concentration of silver nanoparticles was measured by broth dilution method, calcium alginate / nano-silver composite hydrogel material was prepared by ion crosslinking method and its composition was analyzed. The bacteriostatic effect of nano-silver composite calcium alginate hydrogel on Staphylococcus aureus was detected by bacteriostasis method and HE staining was used to observe the initial inflammatory response of calcium alginate hydrogel to soft tissue. Micro-CT was used to evaluate the ability of calcium alginate hydrogel to repair bone. All the experiments were repeated three times. The data were analyzed by SPSS 19.0 software. The data were analyzed by univariate ANOVA (one-way ANOVA). Dunnett and Bonferronili were used to compare and analyze the data, if the variance was not uniform, Dunnett's analysis was used to show statistical difference. Results the injectable calcium alginate hydrogel was successfully prepared. The results of degradation test showed that the degradation time of calcium alginate hydrogel at 25 mg/mL concentration was better than that of bone tissue regeneration. All three kinds of calcium alginate hydrogels are in grade 1. Calcium alginate hydrogel and PLA scaffold can promote the expression of inflammatory factors in periodontal ligament cells. Calcium alginate hydrogel promoted the expression of osteoblast-related factors and successfully prepared calcium alginate hydrogel loaded with nano-silver. The composite of nano-silver has bacteriostatic effect, calcium alginate hydrogel causes soft tissue inflammation, and it binds well to the tissue. Conclusion the calcium alginate hydrogel has a better effect on bone tissue defect induction and regeneration than PLA. Conclusion calcium alginate can be successfully constructed and its biological properties in oral tissue have been proved by in vivo and in vitro experiments. The calcium alginate gel with the ability of promoting bone differentiation for 25 mg/mL has the best effect. The calcium alginate hydrogel loaded with silver nanoparticles prepared successfully has bacteriostasis and is helpful to prevent wound infection.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R783.1

【参考文献】